Inhibition of dynamin completely blocks compensatory synaptic vesicle endocytosis

The ability of synapses to sustain signal propagation relies on rapid recycling of transmitter-containing presynaptic vesicles. Clathrin- and dynamin-mediated retrieval of vesicular membrane has an undisputed role in synaptic vesicle recycling. There is also evidence for other modes of vesicle retrieval, including bulk retrieval and the so-called kiss-and-run recycling. Whether dynamin in required for these other modes of synaptic vesicle endocytosis remains unclear. Here, we have tested the role of dynamin in synaptic vesicle endocytosis by using a small molecule called dynasore, which rapidly inhibits the GTPase activity of dynamin with high specificity. Endocytosis after sustained or brief stimuli was completely and reversibly blocked by dynasore in cultured hippocampal neurons expressing the fluorescent tracer synaptopHluorin. By contrast, dynasore had no effect on exocytosis. In the presence of dynasore, low-frequency stimulation led to sustained accumulation of synaptopHluorin and other vesicular proteins on the surface membrane at a rate predicted from net exocytosis. These vesicular components remained on surface membranes even after the stimulus was terminated, suggesting that all endocytic events rely on dynamin during low-frequency activity as well as in the period after it. Ultrastructural analysis revealed a reduction in the density of synaptic vesicles and the presence of endocytic structures only at synapses that were stimulated in the presence of dynasore. In sum, our data indicate that dynamin is essential for all forms of compensatory synaptic vesicle endocytosis including any kiss-and-run events.